The Superior Colliculus-substantia Nigra Pars Compact Neural Circuit And Its Role In The Regulation Of Motor And Anxiety Behavior In Mice

Object:Parkinson’s disease（PD）is the world’s second most common neurodegenerative disease after Alzheimer’s disease.The typical pathological feature of PD is the progressive decrease of dopaminergic neurons（DAN）in substantia nigra pars compacta（SNpc）and the resulting reduced dopamine（DA）release in striatum（Str）.Studies have shown that DAN in SNpc(DAN^SNpc)target the dorsal striatum（dStr）and regulate movement.Recent studies have shown that neural circuit dysfunction may be an important mechanism leading to PD symptoms,but so far,the pathogenesis of PD and the upstream brain regions of SNpc-dStr have not been fully elucidated.Therefore,in this study,the retrograde tracing technique was applied to study the upstream brain regions projecting to SNpc-dStr circuit.The role of superior colliculus（SC）projecting to SNpc-dStr circuit in movement and anxiety behavior of mice was investigated by using anterograde tracing technology,optogenetics and behavioral experiments.Elucidating the upstream brain regions of SNpc-dStr circuit and its effects on movement and anxiety behavior is of great significance for exploring the pathogenesis of PD and seeking new targets for clinical treatment of PD.Methods:1.The retrograde tracing technique was used to explore the upstream brain regions projecting to SNpc-dStr circuit.2.The upstream brain regions of SNpc-DLS circuit（SC）were verified by the anterograde tracing technique and immunofluorescence staining in TH-Cre mice.3.The types of dsRed positive neurons in the SC were identified by immunofluorescence staining.4.Optogenetics was used to activate or inhibit glutamate neurons in SC.Motor ability was detected by open field test（OFT）and anxiety behavior was detected by elevated plus maze（EPM）.Results:1.Retrograde tracing virus r AAV-cre-wpre-p A（retro）was injected in dorsolateral striatum（DLS）and AAV-DIO-EF1α-GFP-TVA and AAV-DIO-EF1α-RVG（1:1 mix）,and retrograde transsynaptic rabies virus RV-Enva-ΔG-dsRed were injected in SNpc.The results show that:dsRed positive neurons were observed in ventromedial striatum（VMS）,nucleus accumbens（NAc）,interstitial nucleus of the posterior limb of the anterior commissure（IPAC）,lateral hypothalamus（LH）, globus pallidum（GP）,ventral pallidum（VP）,internal capsule（ic）, septohypothalamic nucleus（SHy）,nigrostriatal tract（ns）,zona incerta（ZI）,STN,mesencephalic reticular formation（MRF）and SC,suggesting that the above brain regions might be the upstream brain regions of SNpc neurons projecting to DLS.2.AAV-hsyn-Flp was injected into SC of TH-Cre mice and AAV-hsyn-con/fon-Ch R2-EYFP was injected into SNpc by using the anterograde tracing technique.At the same time,immunofluorescence staining was performed to stain Tyrosine hydroxylase（TH）positive neurons in the SNpc.Results showed that the EYFP-positive neurons in SNpc were merged with TH-positive neurons,and the observation in the DLS showed that there were axon endings of EYFP-positive neurons,indicating that the DAN in SNpc projecting to DLS received input from SC.3.The types of dsRed positive neurons in the SC were identified by immunofluorescence staining.The results showed that about 82±1.5%of the SC- dsRed-positive neurons tracked were Vglut positive neurons,indicating that the SC neurons projected into the SNpc-DLS circuit were mainly glutaminergic neurons.4.Optogenetics was used to detect the effects of activating glutamate neurons in SC(Vglut^SC)on motor and anxiety behavior in mice combined with open field test（OFT）and elevated plus maze（EPM）.The results showed that activation of Vglut^SC reduced the total distance（P<0.01）and the speed（P<0.01）of mice in OFT,and the difference was statistically significant.Meanwhile,activation of Vglut^SC reduced the times of entry into the open arm（P<0.001）and the cumulative time in the open arm（P<0.05）,which was statistically significant,These results indicated that activation of Vglut^SCdecreased motor ability and induced anxiety behavior in mice.5.OFT and EPM was used to detect the effects of optogenetic inhibition of Vglut^SC on motor and anxiety behavior in mice.The results showed that inhibition of Vglut^SC reduced the total distance（P<0.01）and the speed（P<0.01）of mice in OFT, and the difference was statistically significant.In the EPM,compared with the control group,inhibition of Vglut^SC had no significant effect on the times into the open arm and the cumulative time in the open arm,indicating that inhibition of Vglut^SC did not induce anxiety behavior in mice.6.Activation of SC glutamatergic neurons projecting to SNpc(Vglut^SC-SNpc)by optogenetics was used to detect its effects on motor and anxiety behavior in mice.The results showed that compared with the control group,activation of Vglut^SC-SNpc had no significant effect on the total distance and speed of mice in OFT.However, the activation of Vglut^SC-SNpcreduced the times of entry into the open arm（P<0.05） and the cumulative time in the open arm（P<0.05）in EPM,and the difference was statistically significant,indicating activation of Vglut^SC-SNpcinduced anxiety behavior in mice.7.Optogenetic technique combined with OFT and EPM were used to investigate the effect of inhibition of Vglut^SC-SNpc on motor and anxiety behavior in mice.The results showed that the inhibition of Vglut^SC-SNpc decreased the total distance（P<0.01）and the speed（P<0.01）of mice in the OFT compared with the control group,and the difference was statistically significant.Inhibition of Vglut^SC-SNpc in EPM decreased the times of entry into the open arm（P<0.01）and the cumulative time in the open arm（P<0.01）,and the difference was statistically significant, indicating inhibition of Vglut^SC-SNpc induced anxiety behavior in mice.Conclusion:In summary,this experiment mainly explored the upstream brain regions projecting to SNpc-DLS circuit,and found VMS,GP,VP,IPAC,ic,ns,LH,ZI,STN,MRF,SC were upstream brain regions of SNpc-DLS circuit,at the same time,it was verified that as the upstream brain region,SC directly projected to the DAN^SNpc-DLScircuit,and the input of SC to the SNpc-DLS circuit was mainly glutaminergic projection.And the results also showed that the activation of Vglut^SC by optogenetics technique decreased the motor ability of mice and induced the anxiety behavior of mice.Inhibition of Vglut^SC only decreased motor performance without affecting anxiety behavior of mice.Activation or inhibition of the Vglut^SC-SNpc also caused anxiety in mice,but only inhibition of Vglut^SC-SNpc reduced motor ability.These results provide a basis for further research on the relationship between SNpc-DLS circuit and PD,as well as exploring new therapeutic targets for PD.